Method of producing recombinant DNA molecules

ABSTRACT

The present invention is directed to an improved method for producing by recombinant methods proteins that occur in nature in two or more subunits; more specifically applicable to proteins that comprise the alpha and beta subunit of FSH.

CROSS-REFERENCE TO PRIOR APPLICATION

This is a U.S. National Phase Application under 35 U.S.C. §371 ofInternational Patent Application No. PCT/EP2004/006600 filed Jun. 18,2004, and claims the benefit of U.S. Provisional Patent Applications No.60/480,581 filed Jun. 20, 2003 and No. 60/493,586 filed Aug. 7, 2003,both of which are incorporated by reference herein. The InternationalApplication was published in English on Dec. 29, 2004 as WO 2004/113565A1 under PCT Article 21(2).

FIELD OF THE INVENTION

The present invention is directed to an improved method for producing byrecombinant methods proteins that occur in nature in two or moresubunits; more generally, it is applicable to the amplification andsubsequent expression of any chimeric DNA molecule that results from theligation of two or more non-contiguous pieces of DNA.

BACKGROUND OF THE INVENTION

The expression of fusion proteins is well known in the art and it isdisclosed for instance in the following patent publications: EP-6694,EP-20290, U.S. Pat. Nos. 4,898,830, 5,452,199, EP-213472, EP-196864,EP-461165.

The manufacture of recombinant proteins of interest (e.g., human) insuitable expression systems is one of the main industrial applicationsof recombinant DNA technology. If, as is often the case, the protein isunstable in the host cell, it may be advantageous to manufacture theprotein of interest in the form of a fusion moiety comprising aprotective (or stabilising) protein which will be subsequently processedat a specific predetermined site in order to free the desired protein.

Another reason for making fusion proteins is to increase expressionlevels and/or to facilitate the purification process by the selection ofsuitable polypeptide sequences and attachment of one or more of suchsequences to the amino- or carboxy-terminal ends of the polypeptide orprotein of interest. A further reason for expressing a fusion proteinmight be that of having the characterizing features of two or moredifferent proteins or subunits in a single chain, thus providing ahigher activity/dose ratio of the protein itself, and/or avoiding theextra steps to obtain ligation of two subunits. One of the classicalproblems associated with the expression of recombinant proteins is thatof obtaining a valuable and reliable source of the nucleic acid to beexpressed.

One way of addressing this problem is to use an mRNA coding for theprotein to be expressed. mRNA is however not always easily found undernatural conditions. For example, in the case of beta-chain humanfollicle stimulating hormone (FSH), the corresponding mRNA can be foundonly in human pituitary cells and only in minute quantities. To beuseable, this mRNA must be taken from the human pituitary cellsimmediately after death.

An alternative is to obtain the coding sequences of interest fromgenomic DNA. This is however a cumbersome and time consuming process asoften, large amounts of unwanted DNA material are present in the initialsample which increases the probability of mutations and other errors.The need therefore still exists for an improved process for generatingnucleic acid sequences to be used for expressing polypeptides, and inparticular for generating nucleic acid sequences to be used forexpression of heterologous recombinant fusion polypeptides.

SUMMARY OF THE INVENTION

The present inventors have now found a method that permits theexpression of proteins and fusion proteins starting from genomic DNAwithout at least one of the foregoing disadvantages associated withknown methods. This new methodology offers a novel approach to theproduction of recombinant proteins: the present methodology makes itpossible to ligate and amplify encoding pieces of DNA at the desiredpositions without the use of restriction enzymes. This means that it ispossible to use only the exact encoding regions of a desired DNA (i.e.without introns) thus avoiding splicing and/or reducing the formation ofnon desired amplicons.

An example of the advantages of this new methodology is represented bythe expression of the human beta FSH. The gene expressing human beta FSHis over 1500 bases long whereas the corresponding coding region is only390 bases long. According to the present methodology it is possible toconstruct an expression construct containing the correct 390 bases DNAsequence without having to use the 1500 bases of the entire gene. It isthus possible to eliminate 1110 extra bases of unwanted and possiblyproblem causing DNA without using restriction enzymes and without goingthrough the extra steps of isolating mRNA, and generating cDNA. Thepresent invention is thus expedient, inexpensive and less error prone.

With the present methodology it is also possible and relatively simpleto design chimeric molecules of DNA expressing the characteristics oftwo different proteins or protein subunits in a single chain or evenexpressing only portions of a particular protein responsible for itsactivity. For example it is possible to express a new protein having FSHactivity as well as LH activity. In other words, it is possible tocreate new proteins with additional, increased or otherwise modifiedactivity. By using small and precise pieces of DNA it is also possibleto manipulate the actual active site of a wild-type protein in order toobtain a smaller polypeptide endowed with the same or similar activity;due to its lower size, the new polypeptide might thus be administered toa patient by different administration routes than the wild-type protein(i.e. by inhalation or transdermally or transmucosally rather than byinjection) or possess other advantages such as increase yield duringrecombinant production of the therapeutic protein.

As will become apparent from the following detailed description and fromthe examples, the present method is based on a series of PCRamplification steps which are initially carried out on twonon-contiguous DNA segments X1 and X2 which are thus fused together. Thethus obtained polynucleotide (X1X2) is then inserted in a suitableexpression vector to express the desired polypeptide in recombinantcells or transgenic animals according to known methods. The polypeptidemay be a fusion polypeptide, or a polypeptide identical to a proteinthat is naturally produced by translation of a cDNA of polynucleotideelements X1 and X2, where X1 and X2 are exon sequences that are notadjacent in the genomic DNA. Additional chimeric polypeptides within thescope of the invention include dual-activity polypeptides, such asluteinizing hormone-follicle stimulating hormone (LH-FSH) or singlepolypeptide, propolypeptide, or prepropolypeptide wherein the matureprotein is LH, FSH, thyroid stimulating hormone (TSH), chorionicgonadotropin (CG), or any other active polypeptide.

In another aspect, the present invention is directed to a chimericfollicle stimulating hormone (FSH) polypeptide having FSH activity butcomprising, instead of two separate polypeptide chains (designated α andβ in the native molecule), a single fusion polynucleotide segmentencoding the α-chain having its 3′ end directly fused to the 5′ end ofthe β-chain. The encoded chimeric polypeptide molecule is termed AB-FSH,and has been shown to possess FSH activity. This method of producing achimeric AB-FSH protein allows for simplified expression of activefollicle stimulating hormone, as the complete protein is encoded in asingle vector and expressed from a single promoter. The method alloesfor easy purification of an active, stable AB-FSH fusion protein, freefrom isolated beta-FSH and/or alpha-FSH chains.

Preferably, AB-FSH is expressed with the signal sequence for the α-chainto direct secretion of the fusion protein out of the expressing cell andfacilitate its subsequent purification. The signal sequence would becleaved off in post-translational modification. Alternately, the signalsequence of the β chain can be used 5′ to the α chain, or the signalsequence of the α chain can be used 5′ to the β chain.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of two pieces of starting material(double-stranded polynucleotide segments) and the associated primersused in the initial PCR reactions to amplify starting materials. PFX1 isa forward PCR primer for Exon 1; PRX1 is a reverse PCR primer for Exon1; “OH” and “HO” represent 3′ terminal hydroxy groups of the doublestranded DNA; PFX2 is a forward PCR primer for Exon 2; and PRX2 is areverse PCR primer for Exon 2.

FIG. 2 is a schematic representation of the PCR step to generate anintermediate PCR product in which a portion of the 5′ end of X2 is addedto the 3′ end of the X1 to make X1UR or a portion of the 3′ end of theX1 is added to the 5′ end of X2 to make DRX2. PRX1-PFX2′ is a reversePCR primer made by combining the PRX1 primer sequence with thecomplementary sequence of the PFX2 primer (PFX2′); and PRX1′-PFX2 is aforward PCR primer made by combining the complementary sequence of thePRX1 primer (PRX1′) with the PFX2 primer sequence.

FIG. 3 is a schematic representation of the third PCR reaction step,which illustrates the steps of denaturation of the intermediate productX1UR and the X2 starting material, followed by annealing to form a mixof Species A and Species B, followed by extension in which Species Aforms X1X2 by 5′-3′ polymerization and Species B forms X1X2 bysimultaneous 5′-3′ polymerization and 5′-3′ exonuclease activity. X1X2is then further amplified in subsequent rounds of denaturation,annealing and extension. Note that an equivalent mechanism would resultin the formation of the X1X2 species from X1 and DRX2 templates usingprimers PFX1 and PRX2.

In FIG. 3, the symbol:

represents Taq DNA polymerase;

indicates the directionality of Taq DNA polymerase processivity; and

and

represent nucleotides released from Species B by the 5′-3′ exonucleaseactivity of Taq DNA polymerase.

FIG. 4 is a photograph of an agarose gel showing the successful PCRamplification of the X1 beta-FSH PCR product (lane 2), the X2 beta-FSHPCR product (lane 3), and the X1X2 beta-FSH PCR product (lane 4). Lane 1is the ØX/Hinc II MK13a (HT Biotechnology Ltd., Cambridge, UK) molecularweight markers.

FIG. 5 is a photograph of an agarose gel showing the successful PCRamplification of the S-FSH-B PCR product (lane 2), the glycalA RT-PCRproduct (lane 3), and the AB-FSH (alpha-beta-FSH) PCR product (lane 4).Lane 1 is the ØX/Hinc II MK13a (HT Biotechnology Ltd., Cambridge, UK)molecular weight markers.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

Definitions

“Amplification” of DNA as used herein denotes the use of polymerasechain reaction (PCR) to increase the concentration of a particular DNAsequence within a mixture of DNA sequences. For a description of PCR seeSaiki et al., Science 1988, 239:487.

The term “gene” means a DNA sequence that codes for or corresponds to aparticular sequence of amino acids which comprise all or part of one ormore proteins or enzymes, which DNA sequence may or may not includeregulatory DNA sequences (such as promoter sequences) and untranslatedsequences (such as the 5′ untranslated region, 3′ untranslated region,and introns). Some genes, which are not structural genes, may betranscribed from DNA to RNA, but are not translated into an amino acidsequence.

In discussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofdefining the sequence in the 5′ to 3′ direction along the nontranscribedstrand of DNA (i.e., the strand having a sequence homologous to thetranscribed mRNA, also known as the sense strand or the “forward”strand). The organization of other DNA sequences relative to theparticular double-stranded DNA molecule may be described hereinaccording to the normal convention wherein sequences at the 5′ end ofthe particular double-stranded DNA are “upstream” sequences (UR), andsequences at the 3′ end of the particular double-stranded DNA are“downstream” (DR) sequences. Note however, that the present method isvalid for the amplification of both the reference sense (“forward”)strand and of its complementary antisense (or “reverse”) strand.

In discussing the structure of a particular single strandedpolynucleotide (such as an oligonucleotide PCR primer or an isolatedstrand of a double stranded DNA molecule), sequences are describedherein according to the normal convention of defining the sequence inthe 5′ to 3′ direction, which 5′ end represents the terminal phosphateend of said single stranded polynucleotide and which 3′ end representsthe terminal hydroxy end of said single stranded polynucleotide. Inparticular, a “forward” primer is an oligonucleotide which hybridizes tothe 3′ end of the antisense (or “reverse”) strand in order to direct5′-3′ polymerization of the complementary sense (or “forward” strand).Conversely, a “reverse” primer is an oligonucleotide which hybridizes tothe 3′ end of the sense (or “forward”) strand in order to direct 5′-3′polymerization of the complementary antisense (or “reverse”) strand.

By “expression construct”, “expression vector” or “construct” is meant anucleic acid sequence comprising a target nucleic acid sequence orsequences whose expression is desired, operably linked to sequenceelements which provide for the proper transcription and translation ofthe target nucleic acid sequence(s) within the chosen host cells. Suchsequence elements may include, for example, a promoter, a signalsequence for secretion, and polyadenylation signal. The expressionconstruct”, “expression vector” or “construct” further comprises “vectorsequences”. By “vector sequences” is meant any of several nucleic acidsequences established in the art which have utility in the recombinantDNA technologies of the invention to facilitate the cloning andpropagation of the expression constructs including (but not limited to)plasmids, cosmids, phage vectors, viral vectors, and yeast artificialchromosomes. The expression constructs of the invention can beintroduced into a host cell, so as to transform the host and promoteexpression (e.g. transcription and translation) of the introduced targetnucleic acid sequence.

A “promoter” or “promoter sequence” is a DNA regulatory region capableof binding RNA polymerase in a cell and initiating transcription of adownstream (3′ direction) coding sequence. For purposes of defining thepresent invention, the promoter sequence is bounded at its 3′ terminusby the transcription initiation site and extends upstream (5′ direction)to include the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase. The promoter may be operatively associated with otherexpression control sequences, including enhancer and repressorsequences.

A sequence “encoding” an expression product, such as a RNA, polypeptide,protein, or enzyme, is a nucleotide sequence that, when expressed,results in the production of that RNA, polypeptide, protein, or enzyme,i.e., the nucleotide sequence encodes an amino acid sequence for thatpolypeptide, protein or enzyme. A coding sequence for a protein mayinclude a start codon (usually ATG) and a stop codon.

By “host cell” is meant a cell which has been transfected or transformedwith one or more expression constructs of the invention. Such host cellsinclude prokaryotic and eukaryotic cells. Preferred eukaryotic cells foruse in the present invention are in vitro cultured mammalian cells, suchas COS cells and CHO cells. The term host cells also encompassestransformed cells found in vivo, such as in a transgenic mammal.

By “transfection” or “transformation” is meant the process ofintroducing one or more of the expression constructs of the inventioninto a host cell by any of the methods well established in the art,including (but not limited to) microinjection, electroporation,liposome-mediated transfection, calcium phosphate-mediated transfection,or virus-mediated transfection. A host cell into which an expressionconstruct of the invention has been introduced by transfection ortransformation is “transfected” or “transformed”.

Amplification of the Chimeric Polynucleotide X1X2

The present inventors have discovered a method for amplifying a chimericpolynucleotide (X1X2) comprising two non-contiguous nucleotide segmentsof interest, X1 and X2. In one embodiment, the X1 and X2 sequences arederived from sequences that encode distinct proteins, such that the X1X2sequence encodes a fusion protein. In another embodiment, the X1 and X2sequences are derived from sequences that encode a single protein. Forexample, X1 and X2 may represent the sequences of two exons of a gene,which are not adjacent in the genomic sequence of the gene (e.g. due toseparation by introns or possibly other exons or a portion of an exon),but which are adjacent in the transcribed mRNA of the gene (e.g. due tosplicing to remove intervening sequences). In this case, the method ofthe present invention allows for the direct production of the joinedX1X2 sequence, wherein X2 is immediately 3′ to X1, without the necessityof isolating spliced RNA, making cDNA, or performing complex restrictiondigestion and ligation of genomic DNA sequences.

The two nucleotide segments of interest, X1 and X2, can be obtained froma nucleic acid mixture comprising a nucleic acid molecule which includesX1 and a nucleic acid molecule which includes X2. Alternatively, the twonucleotide segments of interest, X1 and X2, are included in the samenucleic acid molecule but they are not contiguous. For example, thenucleic acid molecule may be genomic DNA. Preferably, the nucleic acidmolecules are be derived from a mammal, particularly a human.

In one embodiment of the present invention, the method comprises fourdifferent PCR reactions:

In the first reaction a first nucleic acid segment X1 is made andamplified with a first primer set, which primer set comprises (i) afirst primer, PFX1 (which stands for “primer forward for X1”) thathybridizes to the 3′ end of one strand of the X1 sequence and (ii) asecond primer, PRX1 (which stands for “primer reverse for X1”) thathybridizes to 3′ end of the complementary strand of the X1 sequence(FIG. 1). The amplified X1 PCR product may be isolated by any suitablemethod (as described, for example, in “Molecular Cloning: A LaboratoryManual.” 2^(nd) Edition. Sambrook, et al. Cold Spring Harbor Laboratory:1989, “A Practical Guide to Molecular Cloning” Perbal: 1984, and“Current Protocols in Molecular Biology” Ausubel, et al., eds. JohnWiley & Sons: 1989; commercially available kits include, e.g., theQIAquick PCR Purification Kit and the QLAEX II Gel Extraction Kit, bothfrom Qiagen)

In the second reaction, a second nucleic acid segment X2 is made andamplified with a second primer set, which second primer set comprises(i) a second forward primer, PFX2, that hybridizes to the 3′ end of onestrand of the X2 sequence and (ii) a second reverse primer, PRX2, thathybridizes to the 3′ end of the complementary strand of the X2 sequence.The amplified X2 product can be isolated or purified using any suitablemethod.

In the third PCR reaction (FIG. 2), an intermediate molecule X1UR orDRX2 is made and amplified. X1UR comprises the entire first nucleic acidsegment of X1 and a relatively small 5′ segment of X2, wherein the 3′end of X1 is fused to the 5′ segment of X2. DRX2 comprises the entirefirst nucleic acid segment of X2 and a relatively small 3′ segment ofX1, wherein the 5′ end of X2 is fused to the 3′ segment of X1. This stepis carried out with a third primer set. For X1UR, the third primer setcomprises (i) PFX1 and (ii) a fusion primer PRX1-PFX2′, which fusionprimer has the nucleotide sequence of PRX1 followed immediately at its3′ end by the sequence of the complement of PFX2 (designated as PFX2′).For DRX2, the third primer set comprises (i) a fusion primer PRX1′-PFX2,which fusion primer has the nucleotide sequence of the complement ofPRX1 (designated as PRX1′) attached to the 5′ end of the forward primerPFX2, and (ii) the primer PRX2. Either intermediate can be isolated orpurified using any suitable method.

In the fourth reaction (FIG. 3), the desired polynucleotide X1X2 isfinally made and amplified. This PCR reaction relies on the fact thatfor many commercially available sources of Taq DNA polymerase, the Taqenzyme possesses both 5′-3′ DNA polymerase activity and 5′-3′exonuclease activity at certain temperatures.

In one embodiment of this fourth reaction, the following reagents areused: the X1UR amplified product, the X2 amplified product and theprimers PFX1 and PRX2. In this reaction, both X1UR and X2 serve astemplates for PCR using primers PFX1 and PRX2. The templates areannealed to from two species of significance here: Species A and SpeciesB.

Species A consists of the forward (5′-3′) strand of X1UR and the reverse(3′-5′) strand of X2, with the two strands annealed via thecomplementary X2-derived sequences of the UR region of X1UR and the 3′end of the reverse strand of X2. Although neither primer binds toSpecies A, the Taq polymerase will extend the 3′ ends of the annealedoverlap region to produce the double stranded species X1X2.

Species B consists of the reverse (3′-5′) strand of X1UR and the forward(5′-3′) strand of X2, with the two strands annealed via thecomplementary X2-derived sequences of the UR region of X1UR and the 5′end of the forward strand of X2. Both primers bind to this species. The3′ ends of the annealed primers are then extended by the 5′-3′polymerase activity of the Taq polymerase. Both strands of the finalX1X2 product will be intact, because as each primer is extended on itstemplate in the 5′-3 direction, the original annealed strand (reverse ofX1UR or forward of X2) is removed by the 5′-3′ exonuclease activity ofthe Taq polymerase. In other words, there will be no “nick” in thestrand where the extension product of PRX2 “meets” the 5′ end of thereverse strand of X1UR because the latter will be removed by the Taqenzyme, while the (newly synthesized) forward strand of X1X2 will serveas a template for the remainder of the extension product of PRX2 to bebuilt. The same process is at work to result in the full forward strandof X1X2: when the extension product of primer PRX1 “meets” the 5′ end ofthe forward strand of X2, this strand is removed by the Taq enzyme,while the (newly synthesized) reverse strand of X1X2 will serve as atemplate for the remainder of the extension product of PFX1 to be built.

In an alternate embodiment of this fourth reaction, the followingreagents are used: the DRX2 amplified product, the X1 amplified product,and the primers PFX1 and PRX2. In this reaction, both DRX2 and X1 serveas templates for PCR using primers PFX1 and PRX2. This version of thefourth PCR reaction, wherein annealing to form Species A and Species Bis mediated by the complementary X1-sequences of X1 and the DR region ofDRX2, is analogous to the embodiment described immediately above andalso encompassed by the present specification.

In yet another embodiment of this fourth reaction, the followingreagents are used: the X1UR amplified product, the DRX2 amplifiedproduct, and the primers PFX1 and PRX2. In this reaction, both X1UR andDRX2 serve as templates for PCR using primers PFX1 and PRX2. Thisversion of the fourth PCR reaction, wherein annealing to form Species Aand Species B is mediated both by the complementary X2-sequences of theUR region of X1UR and the 5′ end of the forward strand of X2 and by thethe complementary X1-sequences of X1 and the DR region of DRX2, isanalogous to the embodiment described immediately above and alsoencompassed by the present specification.

It will be understood that for extension of Species A no oligonucleotideprimers are necessary. Therefore, the forgoing directions to employ apolymerase that also acts as an exonuclease pertain to amplification ofSpecies B. In practice, however, both extension of Species A and SpeciesB will take place in the same reaction mixture for reasons of efficiencyand convenience, so the same enzyme will be used for both.

In other embodiments of the present invention, the method comprisesfewer than four different PCR reactions.

For example, the present method does not require that both (or all) ofthe starting DNA segments X1 and X2 be PCR amplified or isolated.Therefore, the intermediate products X1UR and/or DRX2 may be generateddirectly from the primary template sequences (e.g. genomic DNA or cDNA)without first performing a PCR to generate X1 or X2. For example, theprimer PFX1 and the hybrid primer PRX1-PFX2′ may be used to amplify X1URdirectly from genomic DNA. Similarly, the primer PRX2 and the hybridprimer PRX1′-PFX2 may be used to amplify DRX2 directly from genomic DNA.Starting from amplified or isolated X1 and X2 sequences, however, makesthe subsequent amplification steps more efficient.

The segment X2 should be amplified or isolated when it is combined withX1UR to assemble the chimeric DNA molecule X1X2. Similarly, the segmentX1 should be amplified or isolated when it is combined with DRX2 toassemble the chimeric DNA molecule X1X2.

According to the preferred embodiment (hereinafter referred to as“Strategy #2”), the present invention is directed to a method for makinga polynucleotide (X1X2) comprising two nucleotide segments of interest,X1 and X2, wherein X2 in X1X2 is immediately 3′ to X1, from a nucleicacid molecule including X1 and the same or a different nucleic acidmolecule including X2, wherein if X1 and X2 originate on the samemolecule, they are not contiguous, the method comprising:

-   -   (a) amplifying a first double stranded nucleic acid segment X1,        which segment comprises a sense and an antisense nucleic acid        strand, with a first primer set, which primer set comprises (i)        a forward primer, PFX1, which hybridizes to the 3′ end of the        antisense strand of X1 and (ii) a reverse primer, PRX1, which        hybridizes to the 3′ end of the sense strand of X1;    -   (b) amplifying a second double stranded nucleic acid segment,        X2, which segment comprises a sense and an antisense nucleic        acid strand, with a second primer set, which second primer set        comprises (i) a forward primer, PFX2, which hybridizes to the 3′        end of the antisense strand of X2 and (ii) a reverse primer,        PRX2, which hybridizes to the 3′ end of the sense strand of X2;    -   (c) isolating the X1 and X2 products of steps (a) and (b)    -   (d) performing PCR in a single reaction vessel, said vessel        comprising the isolated X1 and X2 products of step (c) in        stoichiometric amounts and primers PFX1, PRX2, and a fusion        primer, which fusion primer has the nucleotide sequence of PRX1        preceded at its 5′ end by the sequence of the complement of        PFX2, said PFX2 complement termed PFX2′, wherein PCR performed        in this single vessel results in amplification of an        intermediate double stranded polynucleotide X1UR, which        intermediate comprises a sense and an antisense nucleic acid        strand, and which comprises the double stranded nucleic acid        segment X1 and a 5′ double stranded nucleic acid segment of X2,        wherein the 3′ end of X1 is fused to the 5′ segment of X2, and        wherein said reaction also results in amplification of the X1UR        intermediate to make X1X2 by denaturing and annealing X1UR and        X2 to form annealed species and then extending and amplifying        said annealed species using DNA polymerase possessing both 5′-3′        polymerase activity and 5′-3′ exonuclease activity and primers        PFX1 and PRX2.

Alternatively, step (d) can be carried out by performing PCR in a singlereaction vessel, said vessel comprising the isolated X1 and X2 productsof step (c) in stoichiometric amounts and primers PRX1, PFX2, and afusion primer, which fusion primer has the nucleotide sequence of PFX2preceded at its 5′ end by the sequence of the complement of PRX1, saidPRX1 complement termed PRX1′, wherein PCR performed in this singlevessel results in amplification of an intermediate double strandedpolynucleotide DRX2, which intermediate comprises a sense and anantisense nucleic acid strand, and which comprises the double strandednucleic acid segment X2 and a 3′ double stranded nucleic acid segment ofX1, wherein the 5′ end of X2 is fused to the 3′ segment of X1, and saidreaction also results in amplification of the DRX2 intermediate to makeX1X2 by denaturing and annealing DRX2 and X1 to form annealed speciesand then extending and amplifying said annealed species using DNApolymerase possessing both 5′-3′ polymerase activity and 5′-3′exonuclease activity and primers PRX1 and PFX2.

It will be understood that the method of the present invention may beused to further attach additional sequences (X3) to the chimericmolecule X1X2. For example, where DNA pieces X1X2 and X3 are to beassembled into the polynucleotide X1X2X3, the segment X1X2 would alsohave an extension complementing the 5′ end of X3 if the UR approach isused (as apposed to the DR approach, where X1X2 would be joined to thesegment X3, which has an extension complementing the 3′ end of X1X2).

The foregoing methods are not limited to making nucleic acid sequencesthat encode chimeric proteins, or to assembling nucleic acid sequencesthat encode multi-exon proteins from genomic source DNA. Any two or morepolynucleotide segments can be fused, including restriction fragmentsfrom one or more DNA molecules. The foregoing method can be easilyadapted to produce polynucleotides comprising three or morenoncontiguous or heterologous nucleic acid molecules, or segments ofsuch molecules

Suitable Taq polymerases that have the combined polymerase/exonucleaseactivity are commercially available. See, e.g., Pfu DNA polymerase (#M7741 or # M7745); Tth DNA polymerase (# M2101 or # M2105); Tfl DNApolymerase (# M1945 or # M1945), all available from Promega Corporation,Madison Wis., USA.

The newly amplified polynucleotide of interest X1X2 can thus be isolatedby means of standard methodologies and can be inserted into a suitableexpression system to provide expression of the corresponding fusionpolypeptide.

Expression Constructs

The expression constructs of the invention contain the amplifiedpolynucleotide sequence operably linked to elements necessary for propertranscription and translation of the amplified sequences within thechosen host cells, including a promoter, a translation initiation signal(“start” codon), a translation termination signal (“stop” codon) and apolyadenylation signal. The expression constructs may compriseadditional sequences that modify expression of the amplified sequences,including internal ribosome entry sites (IRES), enhancers, responseelements, suppressors, signal sequences, and the like.

The promoter sequences may be endogenous or heterologous to the hostcell, and may provide ubiquitous (i.e., expression occurs in the absenceof an apparent external stimulus and is not cell-type specific) ortissue-specific (also known as cell-type specific) expression.

Promoters which may be used to control gene expression include, but arenot limited to, cytomegalovirus (CMV) promoter (U.S. Pat. Nos. 5,385,839and 5,168,062), the SV40 early promoter region (Benoist and Chambon,Nature 1981, 290:304-310), the promoter contained in the 3′ longterminal repeat of Rous sarcoma virus (Yamamoto, et al., Cell 1980,22:787-797), the herpes simplex virus (HSV) thymidine kinasepromoter/enhancer (Wagner et al. Proc. Natl. Acad. Sci. USA 1981;82:3567-71), and the herpes simplex virus LAT promoter (Wolfe, et al.Nature Genetics 1992; 1:379-384), the regulatory sequences of themetallothionein gene (Brinster et al., Nature 1982, 296:39-42);prokaryotic expression vectors such as the beta-lactamase promoter(Villa-Komaroff, et al., Proc. Natl. Acad. Sci. USA 1978, 75:3727-3731),or the tac promoter (DeBoer, et al., Proc. Natl. Acad. Sci. USA 1983,80:21-25); see also “Useful proteins from recombinant bacteria” inScientific American 1980, 242:74-94; promoter elements from yeast orother fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase)promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatasepromoter; the human beta-actin promoter (Gunning, et al. Proc. Natl.Acad. Sci USA 1987; 84:4831-4835), the glucocorticoid-inducible promoterpresent in the mouse mammary tumor virus long terminal repeat (MMTV LTR;Klessig, et al. Mol. Cell Biol. 1984; 4:1354-1362), the long terminalrepeat sequences of Moloney murine leukemia virus (MuLV LTR; Weiss, etal. RNA Tumor Viruses. (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.: 1985).

The expression constructs further comprise vector sequences thatfacilitate the cloning and propagation of the expression constructs. Alarge number of vectors, including plasmid and fungal vectors, have beendescribed for replication and/or expression in a variety of eukaryoticand prokaryotic host cells. Standard vectors useful in the currentinvention are well known in the art and include (but are not limited to)plasmids, cosmids, phage vectors, viral vectors, and yeast artificialchromosomes. The vector sequences may contain a replication origin forpropagation in E. coli; the SV40 origin of replication; an ampicillin,neomycin, or puromycin resistance gene for selection in host cells;and/or genes (e.g., dihydrofolate reductase gene) that amplify thedominant selectable marker plus the gene of interest. Prolongedexpression of the encoded target-reporter fusion in in vitro cellculture may be achieved by the use of vectors sequences that allow forautonomous replication of an extrachromosomal construct in mammalianhost cells (e.g., EBNA-1 and oriP from the Epstein-Barr virus).

For example, a plasmid is a common type of vector. A plasmid isgenerally a self-contained molecule of double-stranded DNA, usually ofbacterial origin, that can readily accept additional foreign DNA andwhich can readily be introduced into a suitable host cell. A plasmidvector generally has one or more unique restriction sites suitable forinserting foreign DNA. Examples of plasmids that may be used forexpression in prokaryotic cells include, but are not limited to,pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids,pBTac-derived plasmids, and pUC-derived plasmids.

A number of vectors exist for expression in yeast. For instance, YEP24,YIP5, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehiclesuseful in the introduction of genetic constructs into S. cerevisiae(see, e.g., Broach, et al. “Experimental Manipulation of GeneExpression.” ed. M. Inouye (Academic Press: 1983)). These vectors canreplicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid.

A number of expression vectors exist for expression in mammalian cells.Many of these vectors contain prokaryotic sequences to facilitate thepropagation of the vector in bacteria, and one or more eukaryotictranscription regulatory sequences that cause expression in eukaryoticcells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr,pTk2, pRSVneo, pMSG, pSVT7, pkoneo, and pHyg derived vectors areexamples of mammalian expression vectors suitable for transfection ofeukaryotic cells. Some of these vectors are modified by the addition ofsequences from bacterial plasmids, such as pBR322, to facilitatereplication and drug resistance selection in both prokaryotic andeukaryotic cells. Derivatives of viruses such as the bovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) maybe used for transient expression of proteins in eukaryotic cells. Abaculovirus expression system (see, e.g., “Current Protocols inMolecular Biology.” eds. Ausubel et al. (John Wiley & Sons: 1992)) mayalso be used. Examples of such baculovirus expression systems includepVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derivedvectors (such as pAcUW1), and pBlueBac-derived vectors (such as theβ-gal containing pBlueBac III).

For other suitable expression systems for both prokaryotic andeukaryotic cells, as well as general recombinant procedures, see“Molecular Cloning A Laboratory Manual. 2^(nd) Edition.” Sambrook, etal. (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and 17.

The expression constructs of the invention may be transfected ortransformed into eukaryotic or prokaryotic host cells in vitro.Preferred in vitro host cells are mammalian cell lines, such as COScells and CHO cells. Protocols for in vitro culture of mammalian cellsare well established in the art [see for example, Animal Cell Culture: APractical Approach 3^(rd) Edition. J. Masters, ed. Oxford UniversityPress and Basic Cell Culture 2^(nd) Edition. Davis, J. M. ed. OxfordUniversity Press (2002)]. Techniques for transfection and transformationare well established in the art and may include electroporation,microinjection, liposome-mediated transfection, calciumphosphate-mediated transfection, or virus-mediated transfection [see forexample, Artificial self-assembling systems for gene delivery. Felgner,et al., eds. Oxford University Press (1996); Lebkowski, et al. Mol CellBiol 1988 8(10):3988-3996; “Molecular Cloning: A Laboratory Manual.”2^(nd) Sambrook, et al. Cold Spring Harbor Laboratory: 1989; and“Current Protocols in Molecular Biology” Ausubel, et al., eds. JohnWiley & Sons: 1989).

Production of Biologically Active Chimeric Proteins

According to a preferred aspect of the invention, the present method isused to produce a polynucleotide X1X2 which encodes a biologicallyactive polypeptide E1E2, wherein E1 is the polypeptide sequence encodedby X1 and E2 is the polypeptide sequence encoded by X2.

According to another aspect of the present invention the two nucleotidesegments X1 and X2 are two exons which are both present in the humangenome and wherein each encodes one of two fragments of a biologicallyactive protein.

In one example, X1 and X2 encode the two exons of the beta subunit ofFSH.

In another example, X1 and X2 encode two subunits of a multi-subunitprotein, the alpha-subunit and beta-subunit of FSH. Therefore, accordingto a further aspect of the present invention the polynucleotide (X1X2)encodes a single polypeptide having the activity of chorionicgonadotropin (CG), luteinizing hormone (LH), follicle stimulatinghormone (FSH) or thyroid stimulating hormone (TSH); or encodes a singlepolypeptide having the activity of more than one of these hormones(e.g., activity of both LH and FSH).

Alternatively, the polynucleotide (X1X2) may encode a pre-protein E1E2,wherein the segment E1 represents a fusion partner, as for instance asignal sequence, and E2 is a biologically active mature polypeptide. Thesignal sequence may be the natural signal sequence of E2 or a differentsignal sequence. The objective would be to facilitate secretion of themature E2.

For example we can produce the β subunit of FSH consisting of segmentsX1 and X2. Alternatively, we can produce any other chimeric polypeptideby using heterologous segments of DNA fused together. So the chimericprotein will have properties of both the constituent polypeptides. Newand unique molecules can thus be generated.

As it will be apparent from the examples, which relate to the expressionof the beta-subunit of hFSH and to the expression of a chimeric proteincontaining both the alpha and beta subunits of FSH (termed AB-FSH), themethod according to the present invention can be carried out using anytwo (or more) non contiguous DNA segments. If additional segments areused, additional PCR steps will be necessary, as a person of ordinaryskill will readily appreciate.

EXAMPLES

The present invention is next described by means of the followingexamples. However, the use of these and other examples anywhere in thespecification is illustrative only, and in no way limits the scope andmeaning of the invention or of any exemplified form. Likewise, theinvention is not limited to any particular preferred embodimentsdescribed herein. Indeed, many modifications and variations of theinvention may be apparent to those skilled in the art upon reading thisspecification, and can be made without departing from its spirit andscope. The invention is therefore to be limited only by the terms of theappended claims, along with the full scope of equivalents to which theclaims are entitled.

Example 1 Assembly of Beta-FSH Expression Constructs using a Novel PCRMethodology

The sequence of the genomic DNA encoding the β subunit of human FSH isat Genbank Accession # AH003599. The cDNA sequence of the β subunit ofhuman FSH is SEQ ID NO:1. The signal sequence is depicted as SEQ ID NO:2. Exon 1 has the sequence SEQ ID NO: 3; Exon 2 has the sequence SEQ IDNO: 4. The corresponding amino acid sequence of human β-FSH is at SEQ IDNO: 5. It is to be noted that β FSH exists in various polymorphs ofwhich the present example produces only one. However, any otherpolymorph could have been easily produced by the present method.

A PCR Amplification of Beta-FSH Encoding Sequences

1. Isolation and Amplification of Two DNA Segments Each Encoding One ofTwo Exons of Beta-FSH

Human genomic DNA was extracted from 50 μl of total blood using theNucleo Spin Blood Quick Pure kit (Macherey-Nagel GmBH& Co.). Theisolated DNA was then dissolved in 100 μl of TB Buffer (DNA solution).10 μl of this DNA solution was used as template for two independent PCRreactions (5 μl genomic DNA solution per PCR reaction).

The first PCR reaction was performed using primers specific for exon 1of beta-FSH. The forward primer PFX1 with the sequence (SEQ ID NO: 7)5′-ATG AAG ACA CTC CAG TTT TTC TTC C-3′ corresponds to a segment fromposition 40 to position 64 in SEQ ID NO: 1. The reverse primer PRX1 withthe sequence (SEQ ID NO: 8) 5′-CCT GGT GTA GCA GTA GCC AGC-3′corresponds to the complement of the segment from position 198 toposition 178 of SEQ ID NO: 1. This PCR reaction amplifies the productX1, as illustrated at FIG. 1, left scheme.

The second PCR reaction was performed using primers specific for exon 2of beta-FSH. The forward primer PFX2 with the sequence (SEQ ID NO: 9)5′-GAT CTG GTG TAT AAG GAC CCA-3′ corresponds to the segment fromposition 199 to position 219 of SEQ ID NO: 1. The reverse primer PRX2with the sequence (SEQ ID NO: 10) 5′-TTA TTC TTT CAT TTC ACC AAA GG-3′corresponds to the complement of the segment from position 429 toposition 407 of SEQ ID NO: 1. This PCR reaction amplifies the productX2, as illustrated at FIG. 1, left scheme.

Each independent PCR reaction contained 5 μl human genomic DNA solutionas the template, and the following additional reagents:

-   -   1. 10 μl 10×PCR Buffer (Thermophilic DNA Polymerase 10× Buffer,        magnesium Free M190G, Promega Madison Wis., USA)    -   2. 10 μl 25 mmol MgCl₂    -   3. 5 μl dNTPs stock solution containing 10 μmol each of dATP,        dTTP, dCTP, and dGTP.    -   4. 1 μl of 100 pmol/μl forward primer stock dilution    -   5. 1 μl of 100 pmol/μl reverse primer stock dilution    -   6. 2 μl TAQ (5 U/μl) (Taq DNA Polymerase in Storage Buffer A,        Cat No. Mi865, Promega Madison Wis., USA)    -   7. 65 μl H₂O.

Cycling parameters were as follows: initial denaturation for 2 min at95° C.; 30 cycles of amplification with each cycle consisting of 30 secat 94° C., followed by 30 sec at 50° C., and then 1 min at 72° C.; and afinal extension for 5 min at 72° C. Cycling was performed using aPTC-100™ programmable Thermal Controller (MJ Research Inc, Watertown,Mass., USA).

Amplification of the X1 and X2 products (beta-FSH exon 1 and exon 2segments, respectively) by each PCR was confirmed by agarose gel (2% inTAE buffer) electrophoresis for 30 min at 125 mA using the DNA MolecularWeight Marker ØX/Hinc II MK13a (HT Biotechnology Ltd., Cambridge, UK) ascontrol molecular weight markers. The results are shown in FIG. 4 (seeLanes 2 and 3). The X1 and X2 products of the PCR reaction were thenpurified using the PCR Clean-up Kit (Nucleospin Extract. Cat No.740590.50).

2. PCR Amplification of the X1UR Intermediate Product:

A PCR reaction was performed on the X1 product (prepared as describeabove) as template, using the exon 1 forward primer PFX1 (SEQ ID NO: 7)and a hybrid reverse primer PRX1-PFX2′ having the sequence (SEQ ID NO:11): 5′-TGG GTC CTT ATA CAC CAG ATC CCT GGT GTA GCA GTA GCC AGC-3′,where the sequence of PRX1 is italicized and the reverse complement ofthe sequence of PFX2 is underlined.

The PCR reaction mixture contained 1 μl X1 PCR product (exon 1 ofbeta-FSH: SEQ ID NO: 3) solution as the template, and the additionalreagents listed above. Cycling parameters were as follows: initialdenaturation for 2 min at 95° C.; 30 cycles of amplification with eachcycle consisting of 30 sec at 94° C., followed by 1 min at 50° C., andthen 2 min at 68° C.; and a final extension for 10 min at 72° C. Cyclingwas performed using a PTC-100™ programmable Thermal Controller (MJResearch Inc, Watertown, Mass., USA). This reaction is illustrated atFIG. 2, top scheme.

Amplification of the X1UR intermediate product by PCR was confirmed byagarose gel (2% in TAE buffer) electrophoresis for 30 min at 125 mAusing the DNA Molecular Weight Marker ØX/Hinc II MK13a (HT BiotechnologyLtd., Cambridge, UK) as control molecular weight markers. The X1UR PCRproduct was then purified using the PCR Clean-up Kit (NucleospinExtract. Cat No. 740590.50) to yield pure DNA in a final volume of 5 μl.Purity and size of the DNA was confirmed by agarose (2% in TAE buffer)electrophoresis for 30 min at 125 mA using the DNA Molecular WeightMarker ØX/Hinc II MK13a (HT Biotechnology Ltd., Cambridge, UK) ascontrol molecular weight markers.

The intermediate X1UR beta-FSH PCR product (SEQ ID NO: 13) consists ofthe X1 (beta-FSH exon 1; SEQ ID NO:3) sequence extended at its 3′ end bythe first 21 base pairs of SEQ ID NO:4 (the beta-FSH exon X2 sequence).

3. PCR Amplification of the X1X2 (Exon 1-Exon 2 Beta-FSH) Product

Two different strategies were employed to amplify the product X1X2.

Strategy #1:

In the first strategy, illustrated at FIG. 3, a PCR reaction wasperformed using the exon 1 forward primer PFX1 (SEQ ID NO: 7) and a theexon 2 reverse primer PRX2 (SEQ ID NO: 10). The earlier amplifiedproducts X1UR and X2 were the templates for this reaction. This PCRreaction contained the following reagents:

-   -   1. 10 μl 10×PCR Buffer (Thermophilic DNA Polymerase 10× Buffer,        magnesium Free M190G, Promega Madison Wis., USA)    -   2. 10 μl 25 mmol MgCl₂    -   3. 5 μl dNTPs stock solution containing 10 μmol each of dATP,        dTTP, dCTP, and dGTP.    -   4. 1 μl X1UR intermediate PCR product (SEQ ID NO: 13)    -   5. 1 μl X2 PCR product (exon 2 of beta-FSH: SEQ ID NO: 4)    -   6. 1 μl of 100 pmol/μl PFX1 (beta-FSH exon 1 forward primer (SEQ        ID NO: 7)) stock dilution    -   7. 1 μl of 100 pmol/μl PRX2 (beta-FSH exon 2 reverse primer (SEQ        ID NO: 10)) stock dilution    -   8. 2 μl TAQ (5 U/μl) (Taq DNA Polymerase in Storage Buffer A,        Cat No. Mi865, Promega Madison Wis., USA)    -   9. 65 μl H₂O

Cycling parameters were as follows: initial denaturation for 2 min at95° C.; 30 cycles of amplification with each cycle consisting of 30 secat 94° C., followed by 1 min at 50° C., and then 2 min at 68° C.; and afinal extension for 10 min at 72° C. Cycling was performed using aPTC-100™ programmable Thermal Controller (MJ Research Inc, Watertown,Mass., USA).

Amplification of the X1X2 product by this PCR reaction was confirmed byagarose (2% in TAE buffer) electrophoresis performed as described above.The results are shown in FIG. 4 (see Lane 4). The product of this PCRreaction was then purified using the PCR Clean-up Kit (NucleospinExtract. Cat No. 740590.50) to yield pure DNA in a final volume of 50μl. Purity and size of the DNA was confirmed by agarose (2% in TAEbuffer) electrophoresis performed as described above.

The size of the product was determined by agarose gel electrophoresis(performed as described above) to be about 390 bp. The sequence of thisproduct was confirmed to be the full length coding sequence forbeta-FSH, X1X2 (SEQ ID NO: 6; see Genbank Accession # NM_(—)000510), byconventional sequence analysis using the primer PFX1 (beta-FSH exon 1forward primer (SEQ ID NO: 7)).

Strategy #2:

In an alternate strategy the final X1X2 PCR product was generated by asingle three primer PCR using the X1 and X2 products as template. Thisstrategy shortened the cloning process by one PCR step. In thisstrategy, the primers PRX2, PFX1, and the hybrid reverse primerPRX1-PFX2′ were used on the X1 and X2 products as template, therebyamplifying first the X1UR intermediate and then the X1X2 final productin a single PCR reaction. The PCR reaction contained the followingreagents:

-   -   1. 10 μl 10×PCR Buffer (Thermophilic DNA Polymerase 10× Buffer,        magnesium Free M190G, Promega Madison Wis., USA)    -   2. 10 μl 25 mmol MgCl₂    -   3. 5 μl dNTPs stock solution containing 10 μmol each of dATP,        dTTP, dCTP, and dGTP.    -   4. 2 μl X1 PCR product (SEQ ID NO: 3)    -   5. 2 μl X2 PCR product (exon 2 of beta-FSH: SEQ ID NO: 4)    -   6. 1 μl of 100 pmol/μl PRX2 (beta-FSH exon 2 reverse primer (SEQ        ID NO: 10)) stock dilution    -   7. 1 μl of 100 pmol/μl forward primer PFX1 (beta-FSH exon 1        forward primer (SEQ ID NO: 7) stock dilution    -   8. 1 μl of 100 pmol/μl hybrid reverse primer PRX1-PFX2′ (SEQ ID        NO: 11) stock dilution    -   9. 2 μl TAQ (5 U/μl) (Taq DNA Polymerase in Storage Buffer A,        Cat No. Mi865, Promega Madison Wis., USA)    -   10. 65 μl H₂O

Cycling parameters were as follows: initial denaturation for 2 min at95° C.; 30 cycles of amplification with each cycle consisting of 30 secat 94° C., followed by 1 min at 50° C., and then 2 min at 68° C.; and afinal extension for 10 min at 72° C. Cycling was performed using aPTC-100™ programmable Thermal Controller (MJ Research Inc, Watertown,Mass., USA).

Amplification of the X1X2 product by this PCR reaction was confirmed byagarose (2% in TAE buffer) electrophoresis performed as described above.The product of this PCR reaction was then purified using the PCRClean-up Kit (Nucleospin Extract. Cat No. 740590.50) to yield pure DNAin a final volume of 50 μl. Purity and size of the DNA was confirmed byagarose (2% in TAE buffer) electrophoresis performed as described above.

The size of the product was determined by agarose gel electrophoresisperformed as described above) to be about 390 bp. The sequence of thisproduct was confirmed to be the full length coding sequence forbeta-FSH, X1X2 (SEQ ID NO: 6; see Genbank Accession # NM_(—)000510), byconventional sequence analysis using the primer PFX1 (beta-FSH exon 1forward primer (SEQ ID NO: 7)).

Though the result was the same using both PCR strategies, we used theX1X2 product from the Strategy #1 PCR reaction because it yielded ahigher quantity and purity of product DNA. However, the second approachis also viable, and has the advantage of proceeding very fast, althoughit requires stoichiometric amounts of the templates.

4. Addition of Shine-Delgarno and Kozak Sequences to the X1X2 (Exon1-Exon 2 Beta-FSH) Product

A 17 nucleotide sequence was added to the 5′ end of the X1X2(Exon1-Exon2 beta-FSH) PCR product (SEQ ID NO: 6), in order to createShine-Delgarno and Kozak consensus sequences (SDK), which directtranslation of the expressed SDK-X1X2 transcript. The sequence was addedby performing a PCR reaction on the X1X2 PCR product using the primerPRX2, the beta-FSH exon 2 reverse primer (SEQ ID NO: 10); and a newforward primer SDK-PFX1 with the sequence 5′-TCG AAG GAG ATA GAA CCA TGAAGA CAC TCC AGT TTT TCT TCC-3′ (SEQ ID NO: 12), where the Shine-Delgarnoand Kozak consensus sequences are underlined, the sequence of the exon 1forward primer PFX1 (SEQ ID NO: 7) is in italics, and the “start” codonfor translation initiation is in boldface. The PCR reaction contained 1μl X1X2 (Exon1-Exon2 beta-FSH) PCR product as the template, and thefollowing additional reagents:

-   -   1. 10 μl 10×PCR Buffer (Thermophilic DNA Polymerase 10× Buffer,        magnesium Free M190G, Promega Madison Wis., USA)    -   2. 10 μl 25 mmol MgCl₂    -   3. 5 μl dNTPs stock solution containing 10 μmol each of dATP,        dTTP, dCTP, and dGTP.    -   4. 1 μl of 100 pmol/μl SDK-PFX1 primer (SEQ ID NO: 12) stock        dilution    -   5. 1 μl of 100 pmol/μl PRX2 (beta-FSH exon 2 reverse primer (SEQ        ID NO:10)) stock dilution    -   6. 2 μl TAQ (5 U/μl) (Taq DNA Polymerase in Storage Buffer A,        Cat No. Mi865, Promega Madison Wis., USA)    -   7. 65 μl H₂O.

Cycling parameters were as follows: initial denaturation for 2 min at95° C.; 30 cycles of amplification with each cycle consisting of 30 secat 94° C., followed by 1 min at 50° C., and then 2 min at 68° C.; and afinal extension for 10 min at 72° C.

Amplification of the SDK-X1X2 product by this PCR reaction was confirmedby agarose (2% in TAE buffer) electrophoresis performed as describedabove. The product of this PCR reaction was then purified using the PCRClean-up Kit (Nucleospin Extract. Cat No. 740590.50) to yield pure DNAin a final volume of 50 μl. Purity and size of the DNA was confirmed byagarose (2% in TAE buffer) electrophoresis performed as described above.

B. Assembly of Beta-FSH Expression Construct

The SDK-X1X2 PCR product was cloned into the pTARGET™ expression vector(Promega) according to manufacturer's instructions (pTARGET™ MammalianExpression System Technical Manual). This system is based upon ligationdirected by annealing of the 3′ adenine overhangs on each end of a PCRproduct (introduced as a natural consequence of Taq polymerase activity)with 5′ thymine overhangs on a linearized pTARGET™ expression vector.The ligated pTARGET™ vector was transformed into E. coli DH5α.

Transformed E. coli DH5α were cultured on solid medium for 24 hours, andthen 10 bacterial colonies from each transformation were used toinoculate individual liquid medium cultures. Plasmid DNA was extractedfrom cultured liquid medium cultures using the JET Quick PlasmidMiniprep Spin Kit/50 (Genomed GmBH, Wielamdstr Bad Oeynhomsen) accordingto manufacturer's instructions.

Isolated plasmid DNA was then checked for incorporation of the SDK-X1X2beta-FSH insert by PCR. In this PCR, primers PFX1 (beta-FSH exon 1forward primer (SEQ ID NO: 7)) and PRX2 (beta-FSH exon 2 reverse primer(SEQ ID NO: 10)) were used to amplify 1 μl of the purified plasmid DNAtemplate. Successful amplification of the X1X2 fragment confirmedsuccessful cloning of the beta-FSH sequences into the pTARGET vector.The resulting expression construct was named pTPKBFSH (pTARGETcontaining the SDK-X1X2 PCR product insert).

Example 2 Expression of Beta-FSH in Mammalian Cell Culture

The pTPKBFSH (pTargeT containing the SDK-X1X2 beta-FSH insert)expression construct was used for the expression of beta-FSH protein inCOS-7L (Invitrogen, catalog No. 11622016), and CHO-S (Invitrogen,catalog No. 11619012) cell lines. The pTPKBFSH construct was transfectedinto the various cell lines using the LIPOFECTAMINE 2000 (Invitrogen)transfection reagent according to manufacturer's instructions. Theexpression vector pSV-β-Galactosidase (Promega) was included as atransfection control.

Following transfection, the cells were cultured in a selective mediumcontaining 1000 μg/ml Geneticine for 7 days. Geneticine-resistant cellswere transferred in a new culture medium containing Geneticine at 200μg/ml, and cultured to a cell density of 10⁶ cells/ml. The supernatantof this culture was than tested for the presence of the beta-FSH subunitusing the Granulosa Cell Aromatase Bioassay (GAB assay) method forFollicle Stimulating Hormone (Dahl et al. Methods Enzymol 1989;168:414-422) for detecting bioactive human beta-FSH. This assayquantitates FSH activity based upon stimulation of aromatase activity ofgranulosa cells, where aromatase activity is measured byradioimmunoassay quantitation of the production of estrogen from anandrostenedione precursor. Thus this assay is a functional assay whichquantifies the amount of biologically active beta-FSH protein in a testsample.

To derive Granulosa Cells for the GAB assay, intact female Spague-Dawleyrats (21-22 days old) were implanted with silastic capsules (10 mm)containing approximately 10 mg diethylstilbestrol (DES) to stimulategranulosa cell proliferation. Four days later, the animals weresacrificed, and the ovaries removed and decapsulated. Follicles of thedecapsulated ovaries were punctured with 27-gauge hypodermic needles,and the granulosa cells removed into McCoy's 5a medium (Gibco),supplemented with penicillin/streptomycin (100 U/mL of each) and 2 mML-glutamine. The cells were pelleted by low speed centrifugation for 5min, and then washed with fresh medium. Cell viability was estimated bytrypan blue staining of a cell aliquot, followed by cell counting usinga hemacytometer. The cells were then diluted in medium to a final volumeof ˜2000-2400 viable cells/ml.

On the day of the assay, fresh GAB assay medium was prepared. The GABassay medium was: McCoy's 5a medium (Gibco), supplemented withpenicillin/streptomycin (100 U/mL of each) and 2 mM L-glutamine, plus1.25 μM androstenedione (Sigma), 0.125 μM diethylstilbesterol (Sigma);37.5 ng/mL human chorionic gonadotropin (Sigma: catalog # C-0434), 0.156mM 1-methyl-3-isobutylxanthine (Sigma), and 1.25 μg/ml insulin (Sigma:catalog # I-1507).

Then 400 μl of GAB assay medium and 60 μL of granulosa cell suspension(˜50,000-80,000 viable cells) were added to each well of a 24-wellplate. Next, 40 μL of pTPKBFSH-transfected COS or CHO cell supernatantor of various concentrations of positive control recombinant FSH(Organon) were added to each well. The plates were then cultured for 3days at 37° C. in a humidified 5% CO₂ incubator. The supernatant fromeach well was then harvested, and 10-20 μl of the supernatant assayedfor estrogen levels by radioimunnoassay. Estrogen radioimmunoassay wasperformed using the Spectria RIA kit for estradiol measurement (OrionDiagnostic, Finland), according to the manufacturer's instructions.

For this assay, various concentrations of the positive controlrecombinant FSH (Organon) were tested and used to calculate a titrationcurve for FSH activity (recombinant FSH concentration in milliUnits permilliliter, mU/ml, versus estrogen radioimmunoassay values, see Table1). This control curve was then used to interpolate the amount ofbeta-FSH activity in the supernatant of pTPKBFSH-transfected COS or CHOcells.

When a 1/1000 dilution of the supernatant from pTPKBFSH-transfectedcells was tested in the granulosa assay, 348 pg/ml of estradiol wasproduced. This value is roughly equivalent to that seen with 25mU/ml ofcontrol recombinant FSH. After correcting for the dilution factor of thetested supernatant, this result indicates that the pTPKBFSH-transfectedcell lines produce bioactive beta-FSH at a quantity of about 25Units/ml.

TABLE 1 Calibration curve values for FSH activity Concentration of 25 50100 200 400 recombinant FSH tested (mU/mL*) Concentration of 322 7101900 3400 4100 estradiol produced (pg/ml) *According to the manufacturer(Organon), recombinant FSH shows an activity profile of approximately8000 Units per mg of total recombinant protein.

Example 3 Production and Expression of Chimeric DNA for AB FSH

The human FSH hormone protein is multi-subunit protein composed of analpha subunit plus a beta subunit. The complete cDNA sequence for thealpha subunit is SEQ ID NO: 14 (Genbank Accession number NM_(—)000735).This example describes the production of an active human FSH protein inwhich both subunits of the hormone are contained in a single proteinchain.

A. Beta-FSH Subunit Encoding Nucleic Acid Sequence

The beta-FSH subunit encoding sequence (X1X2; SEQ ID NO: 6) was preparedas described in Example 1.

B. Alpha-FSH Subunit Encoding Nucleic Acid Sequence

mRNA was isolated from human placental tissue using the Oligotex DirectmRNA Mini Kit (Qiagen, Cat. No. 72022) according to manufacturer'sinstructions. 5 μl of the isolated mRNA was used as template in areverse transcriptase-polymerase chain reaction (RT-PCR), using theprimers HCG-SENT (5′-ATG GAT TAC TAC AGA AAA TAT GCA GCT ATC-3′; SEQ IDNO: 15) and HCG-ANTISENT (5′-TTA AGA TTT GTG ATA ATA ACA AGT ACT GCAG-3′; SEQ ID NO: 16). This RT-PCR reaction was performed using theRT-PCR Kit (Gibco) according to manufacture's instructions. The productof this RT-PCR was named glycalA, and confirmed to represent thecomplete coding sequence of human alpha-FSH (SEQ ID NO: 17) by 2%agarose gel electrophoresis (performed as described above; see FIG. 5,lane 3) and sequencing performed using the primers HCG-SENT andHCG-ANTISENT.

The glycalA alpha-FSH RT-PCR product was then cloned into the pLenti6/V5D-TOPO vector using the pLenti6/V5 Directional TOPO cloning kitaccording to manufacturer's instructions (Invitrogen K4955-10). Tofacilitate this cloning, the nucleotide sequence CACC was added to the5′ end of the glycalA RT-PCR product via PCR using the primersHCG-SENTCACC (5′-CAC CAT GGA TTA CTA CAG AAA ATA TGC AGC TAT C-3′ SEQ IDNO: 18) and HCG-ANTISENT (SEQ ID NO: 16). This PCR contained μl glycalA(alpha-FSH) RT-PCR product as the template, and the following additionalreagents:

-   -   1. 10 μl 10×PCR Buffer (Thermophilic DNA Polymerase 10× Buffer,        magnesium Free M190G, Promega Madison Wis., USA)    -   2. 10 μl 25 mmol MgCl₂    -   3. 5 μl dNTPs stock solution containing 10 μmol each of dATP,        dTTP, dCTP, and dGTP.    -   4. 1 μl of 100 pmol/μl primer HCG-SENTCACC (SEQ ID NO: 18) stock        dilution    -   5. 1 μl of 100 pmol/μl primer HCG-ANTISENT (SEQ ID NO: 16) stock        dilution    -   6. 2 μl TAQ (5 U/μl) (Taq DNA Polymerase in Storage Buffer A,        Cat No. Mi865, Promega Madison Wis., USA)    -   7. 65 μl H₂O.

Cycling parameters were as follows: initial denaturation for 2 min at95° C.; 30 cycles of amplification with each cycle consisting of 30 secat 94° C., followed by 1 min at 50° C., and then 2 min at 68° C.; and afinal extension for 10 min at 72° C.

This PCR product, termed CACCglycalA (SEQ ID NO: 19), was then insertedinto the pLenti6/V5 D-TOPO vector according to manufacturer'sinstructions. This expression construct was named pLenti6/V5-glycalA.

This construct can be used for transient expression of the alpha-FSHsubunit in in vitro cell culture. Alternatively, where stable expressionof the alpha-FSH is desired, the pLenti6/V5-glycalA is transfected intothe ViraPower 293FT- Producer cell line, along with the ViralPowerpackaging mix, to generate a packaged lentivirus (according tomanufacturer's instructions: pLenti6/V5 Directional TOPO cloning kit,Invitrogen K4955-10). This packaged retroviruse is used to transduce amammalian cell line in culture, producing a mammalian cell line thatstably expresses the alpha-FSH subunit.

C. Creation of a Nucleic Acid Sequence Encoding Alpha-FSH+Beta-FSH(AB-FSH)

The alpha-FSH and beta-FSH encoding sequences described above were usedto create a chimeric nucleic acid molecule alpha-beta FSH, in which thesequences encoding the mature beta-FSH protein (i.e., no signal peptide)were fused to the 3′ end of sequences encoding the proprotein ofalpha-FSH (i.e., including the signal peptide). The nucleotide sequenceof alpha-beta-FSH (AB-FSH; SEQ ID NO: 20) was created using thePCR-based method described above for the production of X1X2 beta humanFSH. This nucleotide sequence encodes the AB-FSH polypeptide (SEQ ID NO:27).

1. Mature Beta-FSH

A PCR reaction was performed to generate a nucleic acid sequenceencoding mature beta-FSH (i.e., lacking the signal peptide encodingsequences). This PCR reaction was performed using the new forward primerPFMX2 (5′-AAT AGC TGT GAG CTG ACC AA-3′; SEQ ID NO: 21) and the reverseprimer PRX2 (SEQ ID NO: 10) on 1 μl of the purified X1X2 PCR product(see Example 1, above) solution as the template. This reaction containedadditional reagents and was performed using the cycling parameters asdescribed in Example 1A1 (Isolation and amplification of two DNAsegments each encoding one of two exons of beta-FSH) above. Theresulting PCR product was named S-FSH-B (SEQ ID NO: 22), and confirmedby 2% agarose gel electrophoresis (performed as described above; seeFIG. 5, lane 2)

2. Proprotein Alpha-FSH

A second PCR reaction was performed to generate a nucleic acid sequenceencoding the proprotein of alpha-FSH in which the stop codon wasremoved. Removal of the alpha-FSH stop codon is necessary to preventpremature truncation of the alpha-beta-FSH fusion protein. This PCRreaction was performed using the primer HCG-SENT (SEQ ID NO: 15) and thenew primer HCG-ANTISENT/woTAA (5′-AGA TTT GTG ATA ATA ACA AGT ACT GCAGTG G-3′; SEQ ID NO: 23) on the glycalA PCR product (SEQ ID NO: 17) astemplate. This PCR contained 1 μl glycalA (alpha-FSH) PCR product as thetemplate, and the following additional reagents:

-   -   1. 10 μl 10×PCR Buffer (Thermophilic DNA Polymerase 10× Buffer,        magnesium Free M190G, Promega Madison Wis., USA)    -   2. 10 μl 25 mmol MgCl₂    -   3. 5 μl dNTPs stock solution containing 10 μmol each of dATP,        dTTP, dCTP, and dGTP.    -   4. 1 μl of 100 pmol/μl primer HCG-SENT (SEQ ID NO: 15) stock        dilution    -   5. 1 μl of 100 pmol/μl primer HCG-ANTISENT/woTAA (SEQ ID NO: 23)        stock dilution    -   6. 2 μl TAQ (5 U/μl) (Taq DNA Polymerase in Storage Buffer A,        Cat No. Mi865, Promega Madison Wis., USA)    -   7. 65 μl H₂O.

Cycling parameters were as follows: initial denaturation for 2 min at95° C.; 30 cycles of amplification with each cycle consisting of 30 secat 94° C., followed by 1 min at 50° C., and then 2 min at 68° C.; and afinal extension for 10 min at 72° C. The resulting PCR product was namedglycalwoTAA (SEQ ID NO: 24; alpha-FSH sequences without the TAA stopcodon).

3. Alpha-Beta-FSH (AB-FSH)

A first PCR was performed to generate an intermediate nucleic acidmolecule in which a short segment of 5′ mature beta-FSH encodingsequence (S-FSH-B) was linked to the 3′ end of the alpha-FSH sequencewith the stop codon removed (glycalwoTAA). This PCR was performed usingthe glycalwoTAA PCR product as template, the forward primer HCG-SENT(SEQ ID NO: 15), and the hybrid reverse primer ABLIGATION (5′-TTG GTCAGC TCA CAG CTA TTA GAT TTG TGA TAA TAA CAA GTA CTG CAG TGG-3′; SEQ IDNO: 25), where the underlined sequence is the reverse complement ofprimer PFMX2 (SEQ ID NO: 21) and the sequence of primer ANTISENT/woTAA(SEQ ID NO: 23) is in italics. This PCR contained 1 μl glycalwoTAA PCRproduct as the template, and the following additional reagents:

-   -   1. 10 μl 10×PCR Buffer (Thermophilic DNA Polymerase 10× Buffer,        magnesium Free M190G, Promega Madison Wis., USA)    -   2. 10 μl 25 mmol MgCl₂    -   3. 5 μl dNTPs stock solution containing 10 μmol each of dATP,        dTTP, dCTP, and dGTP.    -   4. 1 μl of 100 pmol/μl primer HCG-SENT (SEQ ID NO: 15) stock        dilution    -   5. 1 μl of 100 pmol/μl primer ABLIGATION (SEQ ID NO: 25) stock        dilution    -   6. 2 μl TAQ (5 U/μl) (Taq DNA Polymerase in Storage Buffer A,        Cat No. Mi865, Promega Madison Wis., USA)    -   7. 65 μl H₂O.

Cycling parameters were as follows: initial denaturation for 2 min at95° C.; 30 cycles of amplification with each cycle consisting of 30 secat 94° C., followed by 1 min at 50° C., and then 2 min at 68° C.; and afinal extension for 10 min at 72° C. We named the product of this PCRReaction glycalwoTAAUR (SEQ ID NO: 26).

A second PCR reaction was then performed to generate the finalalpha-beta-FSH sequence (AB-FSH) using the glycalwoTAAUR intermediatePCR product (SEQ ID NO: 26) and the S-FSH-B PCR product (SEQ ID NO: 22)as templates. The PCR was performed using the forward primer HCG-SENT(SEQ ID NO: 15) and the reverse primer PRX2 (SEQ ID NO: 10). This PCRreaction contained the following reagents:

-   -   1. 10 μl 10×PCR Buffer (Thermophilic DNA Polymerase 10× Buffer,        magnesium Free M190G, Promega Madison Wis., USA)    -   2. 10 μl 25 mmol MgCl₂    -   3. 5 μl dNTPs stock solution containing 10 μmol each of dATP,        dTTP, dCTP, and dGTP.    -   4. 1 μl glycalwoTAAUR intermediate PCR product (SEQ ID NO: 26)    -   5. 1 μl X2 S-FSH-B PCR product (SEQ ID NO: 22)    -   6. 1 μl of 100 pmol/μl primer HCG-SENT (SEQ ID NO: 15) stock        dilution    -   7. 1 μl of 100 pmol/μl primer PRX2 (SEQ ID NO: 10) stock        dilution    -   8. 2 μl TAQ (5 U/μl) (Taq DNA Polymerase in Storage Buffer A,        Cat No. Mi865, Promega Madison Wis., USA)    -   9. 65 μl H₂O

Cycling parameters were as follows: initial denaturation for 2 min at95° C.; 30 cycles of amplification with each cycle consisting of 30 secat 94° C., followed by 1 min at 50° C., and then 2 min at 68° C.; and afinal extension for 10 min at 72° C. Cycling was performed using aPTC-100™ programmable Thermal Controller (MJ Research Inc, Watertown,Mass., USA).

The product of this PCR was confirmed to be AB-FSH (SEQ ID NO: 20) by 2%agarose gel electrophoresis (performed as described above; see FIG. 5,lane 4) and by sequencing of the pLenti/AB-FSH construct (see below)using vector primers.

The AB-FSH PCR product was then cloned into the pLenti6/V5 D-TOPO vectorusing the pLenti6/V5 Directional TOPO cloning kit according tomanufacturer's instructions (Invitrogen K4955-10) as described above(Example 3B. “Alpha-FSH subunit encoding nucleic acid sequence”) exceptthat the PCR primers used to add the nucleotide sequence CACC wereHCG-SENTCACC (SEQ ID NO: 18) and PRX2 (SEQ ID NO: 10). This expressionconstruct was named pLenti/AB-FSH.

Example 4 Co-expression of Alpha-FSH and Beta-FSH in Mammalian CellCulture

The pLenti6/V5-glycaLA (pLenti6/V5-D-topo vector containing the glycalAalpha-FSH insert) and the pTPKBFSH (pTargeT containing the SDK-X1X2beta-FSH insert) expression constructs were used for the expression ofthe complete human FSH hormone in COS-7L (Invitrogen, catalog No.11622016), and CHO-S (Invitrogen, catalog No. 11619012) cell lines. Thealpha-FSH and beta-FSH expression constructs were co-transfected intothe various cell lines using the LIPOFECTAMINE 2000 (Invitrogen)transfection reagent according to manufacturer's instructions. Theexpression vector pSV-β-Galactosidase (Promega) was included as atransfection control.

Following transfection, the cells were cultured in a selective mediumcontaining 1000 μg/ml Geneticine and 400 μg/ml Blasticidin for 7 days.Geneticine-Blasticidine-resistant cells were transferred in a newculture medium containing Geneticine (200 μg/ml) and Blasticidin (200μg/mL), and cultured to a cell density of 10⁶ cells/ml. The supernatantof this culture was than tested for the presence of the multisubunit(alpha subunit and beta subunit non-covalently associated) FSH complex.

The presence of the alpha and beta FSH complex was detected using theBIOSOURCE FSH-IRMA Kit (Biosource Europe S.A., Cat # KIP0841-KIP0844),an immunoradiometric assay kit for the quantitation of FollicleStimulating Hormone (FSH). This FSH-IRMA assay is based on the use oftwo monoclonal antibodies, one specific for the alpha subunit of FSH andone for the beta subunit of FSH. Thus this assay only detects thecomplete alpha and beta FSH complex. The supernatant ofpLenti6/V5-glycalA and the pTPKBFSH co-transfected cells was testedusing the FSH-IRMA kit according to manufacturer's instructions. Thisassay indicated that the supernatant contains alpha and beta FSH complexin an amount of 110 U/ml.

Example 5 Expression of the Alpha-beta-FSH Fusion Protein in MammalianCell Culture

The alpha-beta-FSH expression construct pLenti/AB-FSH was transfectedinto COS-7L cells (Invitrogen, catalog No. 11622016) using theLIPOFECTAMINE 2000 (Invitrogen) transfection reagent according tomanufacturer's instructions. The expression vector pSV-β-Galactosidase(Promega) was included as a transfection control.

Following transfection, the cells were cultured in a selective mediumcontaining 400 μg/ml blasticidin (Gibco) for 7 days.Blasticidin-resistant cells were transferred in a new culture mediumcontaining Geneticine at 200 μg/ml, and cultured to a cell density of10⁶ cells/ml. These cells were then harvested and lysed. Cells werelysed by freezing and thawing in 1 mL of culture medium. The lysed cellswere then centrifuged to pellet the cellular debris. The supernatant wasthen harvested and tested for the presence of alpha-beta-FSH.

The presence of the alpha-beta-FSH fusion protein was then detectedusing the BIOSOURCE FSH-IRMA Kit (Biosource Europe S.A., Cat #KIP0841-KIP0844), an immunoradiometric assay kit for the quantitation ofFollicle Stimulating Hormone (FSH). This FSH-IRMA assay is based on theuse of two monoclonal antibodies, one specific for the alfa subunit ofFSH and one for the beta subunit of FSH. The supernatant of lysedpLenti/AB-FSH-transfected COS-7L cells was tested using the FSH-IRMA kitaccording to manufacturer's instructions. This assay indicated that thethe chimeric alpha-beta-FSH protein was present at a concentration of325 U/ml. Note that for this assay, this results indicates the amount ofprotein alpha-beta-FSH protein present, but this assay is not truly afunctional assay.

To verify that the produced alpha-beta-FSH protein is biologicallyactive, the supernatant of lysed pLenti/AB-FSH-transfected COS-7L cellsis tested in the Granulosa assay as described in Example 2, supra. Forthis assay, 400 μl of GAB assay medium and 60 μL of granulosa cellsuspension (˜50,000-80,000 viable cells) are added to each well of a24-well plate. Next, 40 μL of supernatant of lysedpLenti/AB-FSH-transfected COS-7L cells or of various concentrations ofpositive control recombinant FSH (Organon) are added to each well. Theplates are then cultured for 3 days at 37° C. in a humidified 5% CO₂incubator. The supernatant from each well is then harvested, and 10-20μl of the supernatant assayed for estrogen levels by radioimunnoassay.Estrogen radioimmunoassay is performed using the Spectria RIA kit forestradiol measurement (Orion Diagnostic, Finland), according to themanufacturer's instructions.

For this assay, various concentrations of the positive controlrecombinant FSH (Organon) are tested and used to calculate a titrationcurve for FSH activity (recombinant FSH concentration in milliUnits permilliliter, mU/ml, versus estrogen radioimmunoassay values). Thiscontrol curve is then used to interpolate the amount of biologicallyactive alpha-beta-FSH. The alpha-beta-FSH of the invention shows asubstantially higher activity profile in this assay, as expressed inUnits of bioligical activity per mg of protein, than the currentlyavailable recombinant FSH (Organon).

Example 6 Assembly of INF-β/INF-α2B Expression Constructs

The sequence of the genomic DNA encoding the INF-β is at GenbankAccession NM 002167. The cDNA sequence of the INF-β is SEQ ID NO: 28(hereinafter referred to as THIMIOS1) and it corresponds to the abovesequence NM 002167 without the stop codon. The sequence of the matureINF-α2B is a part of Genbank Accession Genbank NM 000605 (hereinafterreferred to as PENNY1).

A PCR Amplification of Beta-FSH Encoding Sequences

1. Isolation and Amplification of Two DNA Segments Each Encoding One ofTwo Interferons (INF-β and INF-α2B)

Human genomic DNA was extracted from 50 μl of total blood using theNucleo Spin Blood Quick Pure kit (Macherey-Nagel GmBH& Co.). Theisolated DNA was then dissolved in 100 μl of TB Buffer (DNA solution).10 μl of this DNA solution was used as template for two independent PCRreactions (5 μl genomic DNA solution per PCR reaction). The first PCRreaction was performed using primers specific for INF-β.

The forward primer has sequence ATGACCAACAAGTGTCTCCTCCAAATTGCT(hereinafter referred to as THIMIOSF). The reverse primer withoutstopcodon has sequence GTTTCGGAGGTAACCTGTAAGTCTGTTAAT (hereinafterreferred to as THIMIOSR). This PCR reaction amplifies the product CDS ofINF-β.

The second PCR reaction was performed using primers specific for themature chain of INF-α2B. The forward primerGACGACGACGACAAGTGTGATCTGCCTCAAACCCA (hereinafter referred to as PENNYF)contains 1-5 prime sequencing expressing an enterokinase site; this sitehas been added to give the expressed protein molecule the option to becut, after production, thus providing, in case, two different products.

The mature INF-α2B sequence containing the enterokinase site(hereinafter referred to as PENNYF) corresponds to SEQ ID NO: 29.

The reverse primer is TCATTCCTTACTTCTTAAAC (hereinafter referred to asPENNYR).

Each independent PCR reaction contained 5 μl human genomic DNA solutionas the template, and the following additional reagents:

-   -   1. 10 μl 10×PCR Buffer (Thermophilic DNA Polymerase 10× Buffer,        magnesium Free M190G, Promega Madison Wis., USA)    -   2. 10 μl 25 mmol MgCl₂    -   3. 5 μl dNTPs stock solution containing 10 μmol each of dATP,        dTTP, dCTP, and dGTP.    -   4. 1 μl of 100 pmol/μl forward primer stock dilution    -   5. 1 μl of 100 pmol/μl reverse primer stock dilution    -   6. 2 μl TAQ (5 U/μl) (Taq DNA Polymerase in Storage Buffer A,        Cat No. Mi865, Promega Madison Wis., USA)    -   7. 65 μl H₂O.

Cycling parameters were as follows: initial denaturation for 2 min at95° C.; 30 cycles of amplification with each cycle consisting of 30 secat 94° C., followed by 30 sec at 50° C., and then 1 min at 72° C.; and afinal extension for 5 min at 72° C. Cycling was performed using aPTC-100™ programmable Thermal Controller (MJ Research Inc, Watertown,Mass., USA).

Amplification of the INF-β and INF-α2B products PCR was confirmed byagarose gel (2% in TAE buffer) electrophoresis for 30 min at 125 mAusing the DNA Molecular Weight Marker øX/Hinc II MK13a (HT BiotechnologyLtd., Cambridge, UK) as control molecular weight markers. The resultsare shown in FIG. 4 (see Lanes 2 and 3). The products of the PCRreaction were then purified using the PCR Clean-up Kit (NucleospinExtract. Cat No. 740590.50).

The thus-obtained INF-β/INF-α2B with enterokinase sitecorresponds to SEQID NO: 30 whereas that without enterokinase sitecorresponds to SEQ IDNO: 31.

In an alternate strategy the final PCR product was generated by a singlethree primer PCR using the INF-β and INF-α2B products as templates and,as PCR primers, THIMIOSF, PENNYR and the ligation primer TINALEME havingthe sequence reported here-below.

ATCACACTTGTCGTCGTCGTTTCGGAGGTAACCTGTAAGTCT

-   -   1. 10 μl 10×PCR Buffer (Thermophilic DNA Polymerase 10× Buffer,        magnesium Free M190G, Promega Madison Wis., USA)    -   2. 10 μl 25 mmol MgCl₂    -   3. 5 μl dNTPs stock solution containing 10 μmol each of dATP,        dTTP, dCTP, and dGTP.    -   4. 2 μl THIMIOS1 PCR product    -   5. 2 μl HESEMESA PCR product    -   6. 1 μl of 100 pmol/μl forward primer stock THIMIOSF    -   7. 1 μl of 100 pmol/μl reverse primer stock PENNYR    -   8. 1 μl of 100 pmol/μl hybrid primer TINALEME    -   9. 2 μl TAQ (5 U/μl) (Taq DNA Polymerase in Storage Buffer A,        Cat No. Mi865, Promega Madison Wis., USA)    -   10. 65 μl H₂O

Cycling parameters were as follows: initial denaturation for 2 min at95° C.; 30 cycles of amplification with each cycle consisting of 30 secat 94° C., followed by 1 min at 50° C., and then 2 min at 68° C.; and afinal extension for 10 min at 72° C. Cycling was performed using aPTC-100™ programmable Thermal Controller (MJ Research Inc, Watertown,Mass., USA).

Amplification of the product by this PCR reaction was confirmed byagarose (2% in TAE buffer) electrophoresis performed as described above.The product of this PCR reaction was then purified using the PCRClean-up Kit (Nucleospin Extract. Cat No. 740590.50) to yield pure DNAin a final volume of 50 μl. Purity and size of the DNA was confirmed byagarose (2% in TAE buffer) electrophoresis performed as described above.The size of the product was determined by agarose gel electrophoresis.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all values are approximate, and areprovided for description.

Patents, patent applications, publications, product descriptions, andprotocols are cited throughout this application, the disclosures ofwhich are incorporated herein by reference in their entireties for allpurposes.

1. A polypeptide comprising the amino acid sequence set forth in SEQ IDNO: 27, wherein the alpha FSH and beta FSH subunits of the of thepolypeptide do not comprise intervening polypeptide sequences.
 2. Apolypeptide comprising an alpha FSH chain and a beta FSH chain, whereinsaid polypeptide comprises the amino acid sequence set forth in SEQ IDNO: 27, wherein said amino acid sequence is encoded by a single fusionpolynucleotide sequence encoding the alpha FSH chain having its 3′ enddirectly fused to the 5′ end of the beta FSH chain.